The overall objectives of the research proposed herein are to obtain a clearer picture of the regulation of cellular Ca ions levels and the role of Ca ions in neurotransmitter release. In collaboration with Dr. Hannah Rehamimoff (Hadassah University, Israel), we have purified an ATP-dependent Ca ions transport system contained in a synaptosomal vesicle fraction from rat brain. To obtain this approximately 100-fold purification, we employed a novel approach developed in this laboratory, termed "transport specificity fractionation." To our knowledge, no comparable degrees of purification of any ion transport protein from the central nervous system has yet been demonstrated. This purification sets the stage for determining the precise molecular mechanism of the transport system's regulation, and in turn how it regulates Ca ions levels in nerve terminals. Such knowledge could contribute to the understanding of secretory mechanisms in general. An immediate goal is to immunocytochemically localize this transport component in brain tissue at the subcellular level. We will accomplish this using monoclonal antibodies to each of the two major protein components (Mr 94,000 and 140,000) in the preparation. This could establish whether the transport system is indeed localized to nerve terminals, and whether the two proteins are localized to separate regions of the terminals. The immunocytochemical localization could establish to what extent the transport component is involved in removal of Ca ions from the nerve terminal vs. the sequestration of Ca ions within it. In parallel, we will determine whether this Ca ions transport system is regulated by the recently discovered factor "calmodulin," by an adenyl cyclase mediated sequence of events, or by some other mechanism. We will also determine the effect of opiates and the endogenous opiate analogues on the activity of this Ca ions transport system in synaptosomes; if a meaningful effect is observed, we can begin to unravel the precise biochemical mechanism of this opiate action.